Our goal is to acquire a more comprehensive understanding of the regulation of human megakaryocytopoiesis. Aspects to be addressed include: (1) determination of the role of macrophages (M) and thymic-derived lymphocytes (T) in modulating megakaryocyte (Meg) development; (2) the effect of isolated mature Meg on the in vitro growth characteristics of Meg progenitor cells; and (3) the mechanism whereby megakaryocytopoiesis is restored in post-cytoreductive cancer chemotherapy. The core methodology will rely on the cloning of human Meg colony forming units (CFU-M) in culture. CFU-M-derived Meg colonies will be identified using an antibody (Ab) probe that recognizes Megspecific glycoproteins and a second fluorescein-labeled Ab directed against the probe Ab.
Our first aim will be approached by deleting or adding M or T lymphocytes, or growth medium conditioned by these cells, to cultures. M will be isolated by adherence to serum-coated petri dishes. T lymphocytes and their helper/suppressor subpopulations will be isolated by cell """"""""panning"""""""" in petri dishes coated with murine monoclonal antibody specific for the various lymphocyte subpopulations. Indirect methods for accomplishing this same purpose will rely on complement-mediated cytotoxicity employing monoclonal antibodies for deleting these cells from the Meg cultures.
The second aim will be pursued utilizing a technique for isolating mature megakaryocytes based on both density and velocity sedimentation. Should an effect be demonstrated, specific megakaryocyte components will be tested to see where the noted effect lies.
Our third aim will be approached by serially studying patients receiving such therapy. The post therapy CFU-M population will be assessed for cloning efficiency and proliferative capacity. Cell suicide techniques employing hydroxyurea and densitometry measurements for quantitating cellular DNA will be carried out to assess DNA synthetic activity and the development of ploidy in recovering megakaryocytes. Production of CFU-M stimulatory factors in the patient's serum will be serially monitored. Primary platelet production problems or those that are intragenically induced may cause serious therapeutic problems in patients with cancer. The anticipated studies may allow this problem to be circumvented or at least alleviated by gathering basic information on human megakaryocytopoiesis and the pertubations chemotherapy imposes on this system. (IS)
| Gewirtz, A M (1995) Megakaryocytopoiesis: the state of the art. Thromb Haemost 74:204-9 |
| Venturelli, D; Lange, B; Narni, F et al. (1988) Prognostic significance of ""short-term"" effects of chemotherapy on MYC and histone H3 mRNA levels in acute leukemia patients. Proc Natl Acad Sci U S A 85:3590-4 |
| Gewirtz, A M (1988) Exogenous and endogenous regulations of human megakaryocytopoiesis. Adv Exp Med Biol 241:149-64 |
| Gewirtz, A M; Xu, W Y; Mangan, K F (1987) Role of natural killer cells, in comparison with T lymphocytes and monocytes, in the regulation of normal human megakaryocytopoiesis in vitro. J Immunol 139:2915-24 |
